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DESCRIPTION:
Ready-to-use colorimetric substrate for caspase-4 and related caspases that recognize the amino acid sequence LEVD. Caspase-4 and related caspase activity can be quantified by spectrophotometric detection of free pNA (= 400 nm) after cleavage from the peptide substrate LEVD-pNA, using a spectrophotometer or multi-well plate reader. The ready-touse caspase substrate provides an economic alternative for large volume users.

ASSAY PROCEDURE:
1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction.
Note: Active recombinant human caspase-4 is available to use as a positive control (BioVision, Cat.# 1084-25, -100).
2. Count cells and pellet 1-5 x 106 cells.
3. Resuspend cells in 50 μl of chilled Cell Lysis Buffer (Cat.# 1067-100) and incubate cells on ice for 10 minutes.
4. Centrifuge for 1 min in a microcentrifuge (10,000 x g).
5. Transfer supernatant (cytosolic extract) to a fresh tube and put on ice.
6. Assay protein concentration.
7. Dilute 100-300 μg protein to 50 μl Cell Lysis Buffer for each assay.
8. Add 50 μl of 2X Reaction Buffer (Cat.# 1068-20, -80) containing 10 mM DTT (Cat.#1201-1) to each sample.
9. Add 5 μl of the 4 mM of LEVD-pNA (200 μM final conc.) and incubate at 37°C for 1-2 hour.
10. Read samples at 400- or 405-nm in a microtiter plate reader, or spectrophotometer using a 100-μl micro quartz cuvette (Sigma), or dilute sample to 1 ml with Dilution Buffer (Cat.# 1066-100, -500) and using regular cuvette (note: Dilution of the samples proportionally decreases the reading).
You may also perform the entire assay directly in a 96-well plate.
Fold-increase in caspase activity can be determined by comparing these results with the level of the uninduced control.
Note: Background reading from cell lysates and buffers should be subtracted from the readings of both induced and the uninduced samples before calculating fold increase in caspase activity.